Catalytic antibodies are antibodies having catalytic activity. Catalytic antibodies may be generated via immunogenesis by immunizing an immune responsive animal with haptens designed as enzyme substrate analogs.
The efficiency of immunogenesis of catalytic antibodies depends in part upon the design of the enzyme substrate analog. Successful generation of catalytic antibodies depends upon an ability to anticipate the mechanism of a chemical reaction. Antibody catalysis employs the energy with which the substrate binds the antibody to lower activation barriers along the reaction coordinate (Lerner, R. A. et al., Science 1991, 252, p. 659-667). Such catalysis depends upon a correct assumption of mechanistic details, i.e., the extent to which the hapten embodies critical features of the rate-determining transition state, and the manner in which the immune system responds to the antigen. Enzymes generate large rate enhancements through the subtle interplay of many favorable binding interactions refined through the process of evolution. Therefore, one way antibody catalysis could be made even more powerful is by similarly harnessing the diversity of the immune response to control the nature and position of amino acid residues in the active site.
Two conventional strategies are employed for designing haptens employable as enzyme substrate analogs, viz.:
1. A strategy employing haptenic analogs of the substrate transition state; and PA0 2. A strategy employing a hapten within a bait and switch strategy.
Conventionally, the designed hapten is employed both to generate the catalytic antibody by administration to an immune responsive animal and to screen the antibody producing hybridomas derived from splenocytes taken from such immune responsive animal.
When haptenic transition state analogs are employed in the screening process, the resultant catalytic antibody is necessarily limited to those antibody which also have binding activity with respect to the haptenic transition state analog. Catalytic antibodies having low avidity or no avidity for the haptenic transition state analog are lost during this screening process. There is reason to believe that such loss may be significant.
If bait and switch haptens are employed, it is difficult to control the location of the charge on the resultant antibody.
An alternative method for obtaining catalytic antibodies and catalytically active proteins employs semisynthetic combinatorial antibody libraries. In this instance the library is generated in vitro and not in vivo. A preferred semisynthetic combinatorial library employs phage displayed antibodies or proteins, i.e., phagemids. This methodology allows selection for desired functionalities within antibody combining sites because large numbers of different proteins are displayed on a phage format thus linking recognition and replication. Phagemid displayed semisynthetic combinatorial libraries are disclosed by Burton, D. R. (Acc. Chem. Res. 1993, 26, p. 405-411), Barbas III, C. F. et al. (Proc. Natl. Acad. Sci. U.S.A. 1992, 89, p. 4457-4461) and Lerner, R. A. et al. (Science 1992, 258, p. 1313-1314). In this way, a single antibody which carries out a chemical reaction can be identified and replicated so long as the chemical event distinguishes the phage-bearing antibody from the rest of the population.
Panning procedures employed for screening antibodies produced by immunogenesis can also be employed for screening semisynthetic antibody libraries displayed on phagemids. Accordingly, binding assays employing substrate transition state analogs and screening protocols employing the bait and switch methodology may be employed for panning antibody libraries displayed on phagemids. However, the limitations of these panning procedures are carried over to the phagemid technology, i.e., many catalytically active antibodies within the phagemid library fail to be identified during the panning process due to low binding affinity with respect to a haptenic substrate analog or due to a failure of the bait and switch methodology.
A major distinction between an immunogenically generated library and a semisynthetic phagemid library is that the semisynthetic phagemid library does not require the use of a hapten. What is needed is a panning methodology employable with semisynthetic phagemid libraries which exploits this difference and which identifies catalytic antibody or catalytic proteins lost by conventional panning protocols.